Open Canalicular System Research Articles

Proteomic analysis of platelet alpha-granules using mass spectrometry.

Platelets have three major types of secretory organelles: lysosomes, dense granules, and alpha-granules. alpha-Granules contain several adhesive proteins involved in hemostasis, as well as glycoproteins involved in inflammation, wound healing, and cell-matrix interactions. This article represents the first effort to define the platelet alpha-granule proteome using mass spectrometry (MS). We prepared a subcellular fraction enriched in intact alpha-granules from human platelets using sucrose gradient ultracentrifugation. alpha-Granule proteins were separated and identified using sodium dodecylsulfate polyacrylamide gel electrophoresis and liquid chromatography-tandem MS. In the sucrose fraction enriched in alpha-granules, we identified 284 non-redundant proteins, 44 of which appear to be new alpha-granule proteins, on the basis of a literature review. Immunoelectron microscopy confirmed the presence of Scamp2, APLP2, ESAM and LAMA5 in platelet alpha-granules for the first time. We identified 65% of the same proteins that were detected in the platelet releasate (J. A. Coppinger et al. [Blood 2004;103: 2096-104]) as well as additional soluble and membrane proteins. Our method provides a suitable tool for analyzing the granule proteome of patients with storage pool deficiencies.

Deficiency of Src homology 2 domain–containing inositol 5-phosphatase 1 affects platelet responses and thrombus growth

Platelets are critical for normal hemostasis. Their deregulation can lead to bleeding or to arterial thrombosis, a primary cause of heart attack and ischemic stroke. Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1) is a 5-phosphatase capable of dephosphorylating the phosphatidylinositol 3,4,5-trisphosphate second messenger into phosphatidylinositol 3,4-bisphosphate. SHIP1 plays a critical role in regulating the level of these 2 lipids in platelets. Using SHIP1-deficient mice, we found that its loss affects platelet aggregation in response to several agonists with minor effects on fibrinogen binding and beta(3) integrin tyrosine phosphorylation. Accordingly, SHIP1-null mice showed defects in arterial thrombus formation in response to a localized laser-induced injury. Moreover, these mice had a prolonged tail bleeding time. Upon stimulation, SHIP1-deficient platelets showed large membrane extensions, abnormalities in the open canalicular system, and a dramatic decrease in close cell-cell contacts. Interestingly, SHIP1 appeared to be required for platelet contractility, thrombus organization, and fibrin clot retraction. These data indicate that SHIP1 is an important element of the platelet signaling machinery to support normal hemostasis. To our knowledge, this is the first report unraveling an important function of SHIP1 in the activation of hematopoietic cells, in contrast to its well-documented role in the negative regulation of lymphocytes.

Platelets as Immune Cells

Beyond an eminent role in hemostasis and thrombosis, platelets are characterized by expert functions in assisting and modulating inflammatory reactions and immune responses. This is achieved by the regulated expression of adhesive and immune receptors on the platelet surface and by the release of a multitude of secretory products including inflammatory mediators and cytokines, which can mediate the interaction with leukocytes and enhance their recruitment. In addition, platelets are characterized by an enormous surface area and open canalicular system, which in concert with specialized recognition receptors may contribute to the engulfment of serum components, antigens, and pathogens. Platelet-dependent increases in leukocyte adhesion may not only account for an exacerbation of atherosclerosis, for arterial repair processes, but also for lymphocyte trafficking during adaptive immunity and host defense. This review compiles a selection of platelet-derived tools for bridging inflammation and vascular disease and highlights the molecular key components governing platelet-mediated mechanisms operative in immune surveillance, vascular remodeling, and atherosclerosis.

Alpha–delta platelet storage pool deficiency in three generations

Alpha–Delta platelet storage pool deficiency (αδ SPD) is a rare inherited bleeding disorder affecting both males and females, occurring in families, as well as sporadically. Patient platelets in most cases are moderately deficient in both α granules and dense bodies. Only one patient has been severely deficient in both organelles. The present study is the first to document a severe decrease in both platelet α granules and dense bodies in four members in three generations of the same family. Efforts to differentiate this disorder from other hypogranular platelets syndromes in the present investigation suggested that the α granules and dense bodies become connected to channels of the open canalicular system (OCS) and lose their contents to the exterior without prior activation of the cells. In contrast, α granule formation in the white platelet syndrome is too slow, and cells leave the bone marrow still in the process of producing organelles. Gray platelet syndrome platelets can make α granules, but their enclosing membranes are unable to retain stored products. As a result, the organelles lose their contents to surrounding cytoplasm in megakaryocytes and platelets, not selectively through the demarcation system channels and OCS channels. Thus, the pathogenesis of αδ SPD is unique.

Tissue factor-enriched vesicles are taken up by platelets and induce platelet aggregation in the presence of factor VIIa

We investigated the interactions of vesicles containing human tissue factor (TF) with platelets and evaluated responses induced by rFVIIa using standard aggregometry, ultrastructural and flow-cytometry techniques. Washed platelets were exposed to a preparation of placental human TF (pTF) or to a relipidated formulation of recombinant human TF (rTF). Under stirring conditions, pTF induced reversible aggregation with platelets returning to their resting state after 5 minutes. This reversible response to pTF was partially inhibited by antibodies against CD62-P, but not by antithrombin agents, and was not observed with rTF. Sequential ultrastructural studies revealed uptake of both TF preparations by platelets involving traffic of vesicles through channels of the open canalicular system (OCS). Immunocytochemical studies on cryosections identified TF in the OCS, and occasionally in the alpha-granules of the platelets. These processes were faster with pTF than with rTF, but both TF preparations accumulated in platelets at the end of incubation periods. Flow cytometry studies revealed the presence of other cellular antigens (CD62-P, CD14 and CD45) associated to the pTF. Addition of rFVIIa to washed platelets exposed to pTF or rTF, caused a thrombin dependent irreversible platelet aggregation. Our studies demonstrate that platelets possess mechanisms to capture and incorporate TF-rich vesicles. These processes are accelerated by the presence of other cellular antigens in the vesicles. Our findings may explain the hemostatic action of rFVIIa in severely hemodiluted patients, but are also relevant for the understanding of potential implications of TF-associated to platelets in the propagation of thrombus.

Platelet pathology in sex-linked GATA-1 dyserythropoietic macrothrombocytopenia II. Cytochemistry

A previous investigation detailed the pathology of platelets in a family with the X-linked GATA-1 G208S mutation causing dyserythropoiesis and megathrombocytopenia. The present study has used ultrastructural immunocytochemistry, cytochemistry, and tannic acid staining to answer questions raised in the original investigation. Earlier studies, as well as ours, had shown that GATA-1 megathrombocytes are hypogranular, but did not definitively determine which organelles are decreased. Cytochemical localization of aryl sulfatase revealed that lysosomes were present in normal numbers, and the whole mount technique showed a normal frequency of dense bodies rich in arlenine nucleotides and serotonin. Thus α granules were the only organelles deficient in GATA-1 platelets. Tannic acid staining confirmed that the membranes wrapped around each other to form tubular inclusions come from elements of the dense tubular system. The unique tubular membrane inclusions in GATA-1 megathrombocytes, thought originally to derive from endoplasmic reticulum in the parent cell, were shown to be in direct continuity with elements of the surface connected open canalicular system (OCS), and to drive from the demarcation membrane system (DMS) of the megakaryocyte. Platelets in platelets and platelets in platelets in platelets were independent cells, and not derived by cytoplasmic sequestration in the enclosing macrothrombocytes. Fully spread GATA-1 platelets incubated with fibrinogen coated gold (Fgn/Au) particles before or after fixation bound as many Fgn/Au particles as normal spread platelets and moved the Fgn/Au- GPIIb/IIIa complexes from peripheral margins to cell centers and into channels of the OCS as efficiently. Exposure of spread normal platelets to bovine vWF resulted in coverage of the surface from edge to edge with multimers detected by anti-vWF antibody and protein A gold. Spread GATA-1 platelets bound very few vWF multimers, which were much smaller in size than those on normal spread cells, but were able to move then to cell centers. These findings support the concept that GATA-1 platelets are macrothrombocytes because they are not able to detach normally from each other during separation from megakaryocyte proplatelets. The marked decrease in the number and abnormal distribution of GPIb/IX receptors may play a role in GATA-1 megathrombocyte formation.

Platelet pathology in carriers of the X-linked GATA-1 macrothrombocytopenia

Previous investigations from our laboratory have characterized the ultrastructural and cytochemical pathology of platelets in male members of a family with X-linked GATA-1 G208S macrothrombocytoenia. A large proportion of their circulating platelets were hypogranular macrothrombocytes, resembling cells from patients with the Gray Platelet Syndrome. However, most of the GATA-1 macrothrombocytes contained some alpha granules, and a small number had as many as are present in normal platelets. GATA-1 macrothrombocytes also contained tubular inclusions formed from elements of the dense tubular system wrapped around each other like scrolls. Many macrothrombocytes contained flat tubular membrane sheets connected to channels of the open canalicular system, platelets in platelets and platelets attached to platelets forming very large macrothrombocytes. The present study has examined one obligate and three potential female carriers in this family. Thin sections of their platelets examined in the electron microscope revealed features consistent with the pathology observed in male family members. Most of their platelets were normal-sized, discoid cells containing the usual complement of alpha and delta storage organelles and channels of the dense tubular system and OCS. However, a significant number of giant platelets containing the usual frequency of alpha and delta granules and hypogranular and agranular giant platelets were observed. The frequency of the macrothrombocytes varied in each of the four women studied, but were present in all. The ability of their platelets to bind multimers of vWF, in contrast to male family members, did not differ from normal controls. Near normal as well as normal platelet counts and the ability of their platelets to bind vWF multimers may protect them from the serious bleeding problems of males with the X-linked GATA-1 G208S mutation. Our findings indicate that obligate female carriers of the GATA-1 gene can be detected by examination of their platelets in the electron microscope and distinguished from the pathology of other giant platelet disorders.

Platelet Pathology in GATA-1 Dyserythropoietic Macrothrombocytopenia.

Germline mutations in the X-linked hematopoietic transcription factor, GATA-1, have been associated with dyserythropoietic anemia (DEA), macrothrombocytopenia (MTP), and congenital erythropoietic porphyria. Recently, morphological features suggestive of the Gray Platelet Syndrome (GPS) were described in a pedigree with a germline R216Q GATA-1 mutation (Blood 106:6a, 2005). The present study has evaluated platelets (Pl) in the electron microscope from members of a previously described pedigree (Blood 98:2681–2688, 2001) with GATA-1 G208S, MTP, and dyserythropoiesis without anemia, for whom detailed platelet morphology studies have not been reported. The presence of a hemizygous GATA-1 mutation was confirmed by conventional fluorescent dye chemistry sequencing of DNA from patient peripheral blood mononuclear cells. Their Pl revealed wide variations in size, shape, internal and external structure. Some normal sized and giant Pl contained usual numbers of alpha granules and dense bodies, while most of the large cells were hypogranular. Some contained no membrane systems or organelles. Others were filled with masses of dense tubular system (DTS) channels, and others contained the tubular inclusions found in the Medich Giant Platelets Disorder (Platelets 15:345–353, 2004). Many of the hypogranular cells contained small vacuoles that may have been enclosing membranes of alpha granules. Except for the tubular inclusions, all of these structural variations are observed in GPS Pl (Am. J. Pathol. 95:445–462, 1979). However, additional striking abnormalities were seen in GATA-1 Pl that are not characteristic of GPS Pl. Many GATA-1 Pl contained unusual, closely associated dense double membranes differing from normal elements of rough endoplasmic reticulum, smooth endoplasmic reticulum, DTS or surface connected open canalicular system (OCS), but resembling channels of the OCS after exposure to EDTA. They were observed previously only in megakaryocytes (Mk) from one family with GATA-1 DEA-MTP (Nat. Genet , 24:266–270, 2000). The dense double membranes were often found in parallel association and, in some examples, isolating areas of cytoplasm. However, the isolated areas were not undergoing autophagic degradation as such areas enclosed by elements of the DTS do in White Platelet Syndrome Pl (Platelets 15:173,2004). Instead, they proved to be another unique feature of GATA-1 platelets, the presence of Pl within Pl. The sequestered cells were usually mature in appearance and often discoid in form with circumferential coils of microtubules. In some GATA-1 Pl there were two Pl within the same cell, and on rare occasions, a Pl within a Pl within a Pl. Pl within Pl have never been observed previously in any human platelet disorder. Another unique feature, related to Pl within Pl, was the frequent Pl to Pl surface attachment of non-activated cells forming large macrothrombocytes with no similarity to aggregates of stimulated normal platelets.Conclusion: The substructural abnormalities of Pl from patients with germline GATA-1 mutations share some overlap with those seen in GPS Pl, but are distinct and unique. The MTP, Pl within Pl and surface Pl to Pl attachments suggest a major defect in the formation and separation of GATA-1 Pl from pro-Pl of the parent Mk, resulting in the MTP characteristic of the disorder.

Gray Platelet Syndrome in a Somalian family

The Gray Platelet Syndrome (GPS) is a rare inherited, hypogranular platelet disorder characterized by virtual absence of alpha granules in bone marrow megakaryocytes and circulating plates. Usually only one member of a family is affected, but families with two or more affected individuals have been reported from France, Australia, and Mexico, and, recently, the United States. The current study has evaluated the first family from East Africa with two affected members, a brother and sister. Neither child has had significant bleeding problems. Electron microscopic studies revealed almost complete absence of alpha granules from their platelets. Instead their platelets were filled with vacuoles similar in size to the missing granules. Dense bodies were normal in number in the girl's platelets, but markedly increased in her brother's cells. Tannic acid staining revealed that very few of the vacuoles were connected to channels of the open canalicular system. As a result, contents of the residual alpha granule vacuoles must leak out of the organelles and diffuse through megakaryocyte and platelet cytoplasm to the outside. The route of escape may differ from other hypogranular platelet syndromes, such as alpha-delta platelet storage pool deficiency.

Why human platelets fail to kill bacteria

Recent investigations have suggested that bacteria are taken up by platelets in the same manner as by polymorphonuclear (PMN) leukocytes and monocytes. The phagocytic vacuole containing the organism is completely separated from the cell exterior and the cytoplasm in order to form a killing chamber. Yet, earlier studies demonstrated that platelets do not kill bacteria. The present work has provided a basis for the lack of platelet bactericidal activity. Platelets incubated with Staphylococcus aureus 502A or RN 450 for intervals up to 2 h can take up organisms into sequestration vacuoles that resemble the phagosomes formed by PMN and monocytes. However, staining with tannic acid which forms an electron dense stain with osmic acid reveals that the phagosomes in PMN and monocytes are completely separated from the cell exterior and the cytoplasm. As a result, they are true killing chambers. The engulfment vacuoles containing bacteria in platelets on the other hand are almost never sealed from the cell exterior. Osmium black reaction product stains the inside of the engulfment vacuole and channels of the open canalicular system connecting it to the cell exterior. Thus platelets do not form the killing chamber phagosomes observed in leukocytes and, as a result, cannot kill bacteria.

Assessment of Platelet Function in Horses: Ultrastructure, flow Cytometry, and Perfusion Techniques

We studied equine platelet function and activation using ultrastructural examination, flow cytometry, and perfusion. The main aim of the study was to evaluate hemostatic mechanisms in horses using these techniques. Ultrastructural observations were done on resting and activated platelets. Flow cytometry was used to evaluate binding of antibodies to major platelet glycoproteins (GPIIb-IIIa, GPIV, and GPIb) and activation-dependent antigens (P-selectin and lysosomal integral membrane protein [LIMP]). Perfusion techniques were used to evaluate the interaction between platelets and damaged subendothelium. Aggregation experiments were done to identify the best agonists for flow cytometry. Ultrastructural observations confirmed that equine platelets lack a developed open canalicular system and that release of granule contents occurs by fusion of adjacent granule membranes that ultimately connect with external membranes. Flow cytometry identified a 2-fold increase in binding of antibodies against GPIIb-IIIa and GPIV after activation. Binding of antibodies against P-selectin and LIMP increased from 2.12 and 1.74% to 15.5 and 11.6%, respectively, in response to thrombin and to 21.86 and 10.50%, respectively, in response to collagen. Annexin V binding increased moderately after activation. Perfusion experiments with citrated blood indicated that equine platelets react more strongly to subendothelium than do human platelets. When blood was anticoagulated with low molecular weight heparin, a marked impairment of platelet interactions was observed. In conclusion, although some differences were observed between human and equine platelet function, some techniques currently used to assess human platelet function may be useful to assess equine platelets.

Tissue Factor Enriched Microvesicles Plus Factor VIIa Induce Platelet Aggregation in the Absence of Coagulation Proteins: Evidence for the Involvement of Internalization and Trafficking Mechanisms.

The favorable action of FVIIa in critically deterred hemostasis is not completely elucidated though it has been suggested that FVIIa could activate platelets through mechanisms independent of TF. Standard aggregometry, ultrastructural, and immunocytochemical methods were combined with other techniques to assess the interaction of human TF (hTF) enriched microvesicles (MVs) and rFVIIa in a system with repeatedly washed platelets in absence of plasma proteins. Sequential analysis of morphology and immunolocalization of TF on platelet cryosections were also performed. Modifications in the expression of activation antigens and exposure of anionic phospholipids (Annexin V binding) were evaluated by flow cytometry. Activation of tyrosine phosphorylation mediated mechanisms was also monitored. Several inhibitory strategies were used to characterize mechanisms involved in these interactions. After exposure to hTF bearing MVs (hTF-MVs), platelets became reversibly activated reaching a maximum peak in the first minute (41 ± 3% maximal aggregation). Ultrastructural studies revealed that hTF-MVs became rapidly internalized by platelets. This process induced changes in platelet shape, organelle centralization and enhanced platelet-platelet interactions, but did not result in the formation of compact aggregates. Immunocytochemical studies at ultrastructural level demonstrated hTF-MVs trafficking from the external membranes to the alpha granules via the open canalicular system. Flow cytometry studies detected a moderate increase in the expression of platelet activation antigens. In the absence of rFVIIa platelets always return to an apparent resting state in 5 min, recovering their discoid shape, though immunocytochemical studies still demonstrate TF presence in platelets. When rFVIIa was present together with hTF-MVs, platelets became irreversibly aggregated (81% ± 6%). Ultrastructural studies confirmed internal contraction, degranulation and formation of perfectly structured platelet aggregates. Flow cytometry confirmed a marked expression of activation dependent antigens and a significant enhancement of annexin V binding which was never detected in studies with hTF-MVs alone. Antibodies to GPIIb-IIIa blocked reversible and irreversible aggregation steps. Heparins or hirudin inhibited the irreversible aggregation that occurred in the presence of rFVIIa, but did not prevent the initial reversible aggregation caused by hTF-MVs. Antibodies to P-selectin reduced the initial reversible activation of platelets in response to hTF-MVs, but did completely block the aggregation that occurred in the presence of rFVIIa. This study demonstrates that platelets internalize TF when it is exposed on MVs and confirms the existence of TF trafficking from external sources into internal platelet storage pools. Our results prove that the complex rFVIIa-hTF activates platelets in the practical absence of plasma proteins. If TF can be stored in platelets, the hypothesis of a direct action of rFVIIa on platelets independent of TF should be reconsidered, since internal pools of TF could be exposed on the surface upon activation. Our studies indirectly suggest that TF stored in platelets could enhance the potential thrombogenic capacity of platelets under special pathologic situations.

Increased Bleeding Tendency in a Patient with Caffey Disease Due to a COL1A1 Mutation and a Defect in Platelet Morphology and Function.

Caffey disease or infantile cortical hyperostosis is characterized by hyperirritability, acute inflammation of soft tissues, and profound alterations of the shape and structure of particularly the long bones. This autosomal dominant disorder in 4 unrelated families is caused by a similar missense mutation (R836C) in the gene for the alpha1 chain of type I collagen (COL1A1; Gentile et al, JCI, 2005). The precise link between this mutation and either the localized inflammation problems or the originally described thrombocytosis and easy bruising associated with Caffey disease (Lorber et al, 1979) was not evidenced. In the present study, we studied the platelets from a 6-year-old girl with Caffey disease due to a de novo COL1A1 R836C mutation because of her increased bleeding tendency. Apart from the typical skeletal deformities she also presented with easy bruising. This COL1A1 mutation modulates normal megakaryopoiesis since the patient presented with a relatively high number of platelets (+/−450.000/mL) and a decreased MPV (7-8 fL). All other hematological parameters were normal. Electron microscopy further revealed platelets with a proliferation of the dense tubular system, a pronounced open canalicular system and a reduced number of often smaller dense granules. Platelet ATP secretion was reduced after stimulation with 5 mg/ml Horm collagen (2 mM: nl 3-7 mM). The PFA100 response with either collagen/epinephrine or collagen/ADP was within the normal range. Aggregation studies were suggestive for a selective impairment of platelet activation to collagen since the patient platelets showed a reduced and retarded response towards Horm Collagen, convulxin and the collagen related peptide (CRP-XL) but a normal aggregation with ADP, U46019 and arachidonic acid. Membrane glycoprotein (GP) profiling by flow cytometry showed a normal antibody binding to integrin beta3, integrin alpha2beta1 and GPVI. Since R to C amino acid substitutions in COL1A1 are associated with an increased disulfide crosslinking within mutant collagen fibers (Gentile et al, JCI, 2005) but also with other cysteine-containing proteins as integrins, we hypothesized that the platelet integrins might be triggered when mature megakaryocytes are in contact with the collagen type I of the extracellular matrix of the patients bone marrow. Immunoblot analysis of platelet lysates from the patient indeed showed the presence of a 190 kDa COL1A1 band, which could not be detected in control samples. In addition, beta1 integrin could be co-immunoprecipitated with an anti-COL1A1 antibody in platelet lysates from the patient. Further studies are needed to determine whether this COL1A1 R836C binding to platelet collagen receptors is responsible for the defective collagen signaling in this patient by receptor desensitization. In conclusion, we here present the first collagen type I mutation that leads to a defective platelet ultra structure and function.

SNARE Protein Distribution in Platelets: Extracellular Localization of SNAP-23 and Syntaxin 2.

SNARE proteins are a family of membrane-associated proteins that mediate granule secretion in many cell types including platelets. These proteins are oriented such that all but the membrane spanning region of the protein resides in the cytosol. Complex formation between SNARE proteins brings granule membranes in close apposition to surface-connected membranes, facilitating membrane fusion and granule release. Initial studies evaluated the subcellular localization of SNARE proteins in resting platelets by electron microscopy using a pre-embedding immunonanogold technique. These studies demonstrated that VAMP 3 is associated primarily with granules, the majority of SNAP-23 is associated with plasma membrane, and syntaxin 2 is distributed relatively equally between granules, plasma membranes, and membranes of the open canalicular system. Following activation of platelets with either the thrombin receptor activating peptide, SFLLRN, or the phorbol ester, PMA, we observed staining of SNAP-23 and syntaxin 2 on the extracellular surface of platelets. VAMP 3 remained intracellular. Extracellular localization of SNARE proteins has not previously been observed in any cell type. We therefore sought to confirm extracellular localization. Flow cytometry of intact platelets using antibodies directed at SNAP-23, syntaxin 2, and VAMP 3 demonstrated extracellular localization of SNAP-23 and syntaxin 2, but not VAMP 3, following activation of platelets with either SFLLRN or PMA. Incubation of intact pacified platelets with trypsin resulted in degradation of SNAP-23 and syntaxin 2 as detected by immunoblotting. In contrast, trypsin failed to cleave the intracellular proteins VAMP 3 or Rab4. SNAP-23 associates with membranes via palmitoyl moieties. Incubation of intact platelets with acyl-protein transferase 1 (APT1), an enzyme that removes palmitate from proteins, resulted in release of SNAP-23 from the platelet membrane. Similarly, incubation of intact platelets with botulinum toxin C, which specifically cleaves certain syntaxin isoforms, released syntaxin 2 from the platelet surface. Of note, APT1 and botulinum toxin released SNAP-23 and syntaxin 2, respectively, from the surface of pacified, resting platelets. These data suggested that SNAP-23 and syntaxin 2 reside on the surface of the resting platelet. We therefore sought to evaluate whether increased surface expression of SNAP-23 and syntaxin 2 following platelet activation results from epitope unmasking of SNARE proteins already present on the platelet surface. Incubation with trypsin prevented PMA-induced stimulation of SNAP-23 and syntaxin 2 expression, but not PMA-induced P-selectin expression. Furthermore, incubation of resting intact platelets with botulinum toxin C resulted in release of SNAP-23, suggesting that SNAP-23 and syntaxin 2 form a complex on the resting platelet surface. These results indicate that enhanced detection of SNARE proteins on the platelet surface following activation results not from translocation of SNARE proteins from internal stores, but rather from activation-induced epitope exposure of SNARE proteins localized to the extracellular surface in resting platelets. These data represent the first demonstration of extracellular SNARE proteins and suggest that SNAP-23 and syntaxin 2 localize to the platelet surface during granulopoiesis.

Interaction of Recombinant Factor VIIa with Rehydrated, Lyophilized Platelets.

The experiments presented here were undertaken to determine if factor VIIa (rFVIIa, the Novo Nordisk product NovoSeven™) will directly bind to rehydrated, lyophilized (RL) platelets (Stasix™ platelets, Entegrion, Inc. trade) for the formation of a catalytic surface with an enhanced ability to generate thrombin. The relationship of rFVIIa to the RL platelet surface was examined by measuring equilibrium and non-equilibrium binding of the coagulation factor to the cells, by studying the subcellular localization of the coagulation factor on RL platelets, and by following the effects of the surface modification on the kinetics of thrombin generation. The association of rFVIIa with RL platelets occurred with an on rate of 3.6x103 sec−1moles−1. Saturation occurred in minutes and was calcium dependent. Disassociation (in plasma or citrated saline) was slow, with over half of the coagulation factor remaining bound after two hours (with slow and fast rate constants of 5.0x10−5 and 4.1x10−4 sec−5 respectively). These results define a binding site with an apparent equilibrium constants of 110 nM. Equilibrium binding of rFVIIa to RL platelets was analyzed with flow cytometry and Western analysis. The rFVIIa was bound to RL platelets in a dose-dependent manner when incubated at concentrations of 0.3 to 10.0 uM rFVIIa and 3x104 to 106 RL platelets/ul in citrated saline. When high concentrations of rFVIIa were bound to RL platelets densities of over one million molecules of rFVIIa per RL platelet was obtained. Fluorescent microscopy analysis revealed that the rFVIIa was localized to the surface membrane and that some rFVIIa localized internally to the outer surface of the surface connected open canalicular system and/or sites of internal trafficking. Flow cytometric analysis with annexin V demonstrated that considerable quantities of phosphatidylserine were present on the external surface of the RL platelet membrane for potential facilitation of rFVIIa binding. The effect of RL platelet surface modification by rFVIIa on thrombin generation was investigated by following the hydrolysis of the thrombin-specific fluorogenic substate D-phe-pro-arg-ANSNHin plasma. rFVIIa and RL platelets accelerated thrombin generation in this system with rFVIIa being approximately twice as effective (per molecule of the recombinant protein) when added to the assay system pre-bound to RL platelets as compared to being initially free in the plasma. Similar results were obtained when free and RL platelet bound rFVIIa were tested in factor IX-deficient plasma. These experiments show that rFVIIa retains activity when super-saturated on the RL platelet membrane. The results of the studies presented here suggest that RL platelets can be used to concentrate rFVIIa at sites of vascular injury.

The platelet receptor for type III collagen (TIIICBP) is present in platelet membrane lipid microdomains (rafts)

Platelet interactions with collagen are orchestrated by the presence or the migration of platelet receptor(s) for collagen into lipid rafts, which are specialized lipid microdomains from the platelet plasma membrane enriched in signalling proteins. Electron microscopy shows that in resting platelets, TIIICBP, a receptor specific for type III collagen, is present on the platelet membrane and associated with the open canalicular system, and redistributes to the platelet membrane upon platelet activation. After platelet lysis by 1% Triton X-100 and the separation of lipid rafts on a discontinuous sucrose gradient, TIIICBP is recovered in lipid raft-containing fractions and Triton X-100 insoluble fractions enriched in cytoskeleton proteins. Platelet aggregation, induced by type III collagen, was inhibited after disruption of the lipid rafts by cholesterol depletion, whereas platelet adhesion under static conditions did not require lipid raft integrity. These results indicate that TIIICBP, a platelet receptor involved in platelet interaction with type III collagen, is localized within platelet lipid rafts where it could interact with other platelet receptors for collagen (GP VI and alpha2beta1 integrin) for efficient platelet activation.

High cystine in platelets from patients with nephropathic cystinosis: a chemical, ultrastructural, and functional evaluation

Aim: To investigate the morphology and function of platelets in nephropathic cystinosis (NC). Methods: Seven patients (mean age, 6.5 years; SD, 20 months) with NC were investigated. Their platelets were...

The actin cytoskeleton differentially regulates platelet α-granule and dense-granule secretion

AbstractStimulation of platelets with strong agonists results in centralization of cytoplasmic organelles and secretion of granules. These observations have led to the supposition that cytoskeletal contraction facilitates granule release by promoting the interaction of granules with one another and with membranes of the open canalicular system. Yet, the influence of the actin cytoskeleton in controlling the membrane fusion events that mediate granule secretion remains largely unknown. To evaluate the role of the actin cytoskeleton in platelet granule secretion, we have assessed the effects of latrunculin A and cytochalasin E on granule secretion. Exposure of platelets to low concentrations of these reagents resulted in acceleration and augmentation of agonist-induced α-granule secretion with comparatively modest effects on dense granule secretion. In contrast, exposure of platelets to high concentrations of latrunculin A inhibited agonist-induced α-granule secretion but stimulated dense granule secretion. Incubation of permeabilized platelets with low concentrations of latrunculin A primed platelets for Ca2+- or guanosine triphosphate (GTP)-γ-S-induced α-granule secretion. Latrunculin A-dependent α-granule secretion was inhibited by antibodies directed at vesicle-associated membrane protein (VAMP), demonstrating that latrunculin A supports soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein-dependent membrane fusion. These results indicate that the actin cytoskeleton interferes with platelet exocytosis and differentially regulates α-granule and dense granule secretion.

Platelets are covercytes, not phagocytes: Uptake of bacteria involves channels of the open canalicular system

Recent studies have suggested that uptake of bacteria by human platelets is more similar to ingestion of the same organisms by neutrophils than previously considered. Platelet pseudopods surround a bacterium by a process of circumferential adherence until the organism is fully enclosed in a vacuole consisting of internalized extracellular space surrounded by plasma membrane. The surface connected, open canalicular system (OCS) is not involved in the process of platelet phagocytosis. The present investigation has reviewed the literature on platelet–particle and platelet–bacterial interaction, and added a new study of the process of bacterial engulfment employing tannic acid as an electron dense tracer. The osmium black reaction product formed by tannic and osmic acids during fixation stained the glycocalyx on the platelet exterior surface and lining channels of the OCS. It also stained the vacuoles containing bacteria in platelets as well as channels of the OCS connected to the engulfment vacuole and exterior surface of the cell. Alpha granules releasing their contents into the OCS and into engulfment vacuoles were also stained by osmium black. The only way that tannic acid could reach and stain the interior surface of engulfment vacuoles during fixation was through patent channels of the OCS. In contrast, neutrophils do not contain an OCS, and phagocytic vacuoles containing organisms are never stained by tannic acid. Thus the platelet is a covercyte, not a phagocyte, and uptake of the bacteria does involve channels of the OCS.

Platelets Are Covercytes, Not Phagocytes.

Youssefian et al. (Blood2002; 99:4021) have recently reported that engulfment of Staphylococcus Aureus (S.A.) by human platelets is more like phagocytosis by polymorphonuclear leukocytes (PMN) than previously considered.1 Electron microsopy and ultrastructural immunocytochemistry revealed that the engulfment vacuoles containing bacteria were entirely made up of invaginated surface membrane. The surface-connected, open canalicular system (OCS), a tortuous system of interconnected channels continuous with the surface membrane, was not involved in uptake of bacteria. Many of our previous investigations have examined platelet-particle and platelet bacterial interaction, and disagree with the findings of Youssefian et al.1 The present study has used tannic acid as an electron dense tracer to evaluate the hypothesis indicating the OCS is not involved in bacterial uptake.1 Platelets in platelet-rich plasma (PRP) or in washed suspensions (WPS) and samples of PMN were incubated at 37°C with S.A. 502A or S.A. RN 450 for 15, 30, 60, and 120 minutes with or without added ADP.1 Fixation of samples was carried out in glutaraldehyde and osmic acid in the presence of tannic acid. The tannic acid selectively binds to the glycocalyx covering the exterior platelet surface and lining channels of the OCS, and converts osmium to osmium black visible as an electron dense stain. It also combines with fibrinogen/fibrin permitting visualization of α-granule secretion into the OCS. Interaction with bacteria during incubation is variable and infrequent, unless the sample is stirred or an aggregating agent like ADP is added.1 The bacterium attaches itself to the platelet surface, often at or near the opening of an OCS channel. The platelet cannot move the bacterium across the surface as it does small particles up to the size of α granules. Instead the platelet molds itself to the surface, just as it does to large latex spheres and flat surfaces. However, the latter surfaces cannot be internalized, and the platelet continues to spread causing evagination of the OCS to add to the total surface area covered. Bacteria can be internalized. Platelet spreading over the organism causes delilation of the openings of OCS channels. More canaliculi are recruited from the spiderweb of interconnected OCS channels to make a vacuole. Tannic acid staining shows the platelet engulfment vacuoles always contain osmium black around the bacterium, whereas PMN phagocytic vacuoles are never stained. α granules secreting fibrinogen/fibrin into OCS channels stained by tannic acid also discharge their contents into engulfment vacuoles containing bacteria. Direct communication between engulfment vacuoles and the platelet exterior through tannic acid stained OCS canaliculi were identified, while communicating OCS channels were out of the plane of section in other examples. Since the only way tannic acid could stain the engulfment vacuoles containing bacteria was by way of the OCS, it is clear that the OCS is involved in their interiorization. Thus the platelet is a covercyte, not a phagocyte, and its interaction with bacteria is very different from that of the PMN which does not have an OCS.